Proteins were separated by SDS Web page and detected with immunoblotting. Chromatin immunoprecipitation Transfected HEK 293 cells, C 1, had been cross linked with 1% formaldehyde in PBS at room temperature for 15 min. Cross linking was carried out with rotation, as well as reaction was stopped by addition of glycine to a ultimate concentration of 125 mM. After two washes with PBS, cells have been lysed in IP buffer and frozen in LN2. Right after thawing the samples were diluted to a last SDS concentration of 0. 1%. Samples were soni cated to produce sheared DNA fragments close to 400 base pairs, and insoluble chromatin was discarded right after centrifugation. Dyna beads ProteinG had been washed with PBS and incubated with antibody at area temperature for 40 min followed by washing with PBS. The soluble chromatin fraction was then additional followed by incubation overnight at 4 C with rotation.
Chromatin equivalent to 200 000 cells was applied per IP with 20 ul Dynabeads ProteinG and two ug antibody, within a complete volume of 1. 2 ml IP buffer. The immunoprecipitates had been washed 5 occasions in IP buffer, prior to DNA was eluted with 1% SDS selelck kinase inhibitor in 100 mM sodium carbonate at 65 C for 10 min. After treatment with RNAse A and proteinase K, cross linking was reversed by incubation at 65 C for eight h. DNA was puri fied using silica columns and eluted in 50 ?l 10 mM Tris HCl. two. 5 ?l of the eluted DNA was employed as template for quantitative real time PCR in a total volume of 20 ?l. Standard curves of genomic DNA had been run alongside the ChIP samples for every primer pair, and analyzed on a LightCycler 480. Input DNA was used to normalize values from ChIP samples. Antibodies For Western immunoblotting the following antibodies were used, rabbit anti HA, mouse anti FLAG M2 antibody, goat anti PIAS1, rabbit anti PIAS1 rabbit anti GFP, mouse anti GAPDH, and mouse anti tubulin.
Anti mouse IgG HRP, anti rabbit IgG HRP, and anti goat IgG HRP were utilised as secondary antibodies. As immunofluores cence antibodies rabbit anti HA, mouse anti FLAG selleckchem M2 antibody, and mouse anti pol II were utilized. Alexa Fluor 488 goat anti rabbit IgG, Alexa 546 goat anti mouse IgM, and Alexa Fluor 633 goat anti mouse IgG1 have been implemented as secondary antibodies. Reporter gene assays CV 1 cells had been plated in 24 very well microplates at a con centration of 2?104 cells per properly the day prior to transfec tion. The cells have been transfected having a total of 0. 8 ug DNA per well. Cells were washed twice in PBS, and lysed in Passive Lysis Buffer 24 hours right after transfec tion. Luciferase exercise was monitored using a Luciferase assay kit. Light emission was established that has a luminometer. Every single experiment was carried out in triplicate, and typical data from 3 inde pendent transfection experiments are presented.